Impact of Protease on Ultraviolet Photodissociation Mass Spectrometry for Bottom-up Proteomics

Recent mass spectrometric studies
have reported enhanced proteome
coverage by employing multiple proteases or by using multiple or alternative
activation methods such as electron-transfer dissociation in combination
with collisional-activated dissociation (CAD). In this study the use
of 193 nm ultraviolet photodissociation for the analysis of thousands
of Halobacterium salinarum peptides generated by
four proteases (trypsin, LysC, GluC, and chymotrypsin) was evaluated
in comparison with higher energy CAD (HCD). Proteins digested by trypsin
resulted in greater sequence coverage for HCD over UVPD. LysC digestion
resulted in similar sequence coverages for UVPD and HCD; however,
for proteins digested by GluC and chymotrypsin 5–10% more sequence
coverage on average was achieved by UVPD. HCD resulted in more peptide
identifications (at 1% false discovery rate) for trypsin (4356 peptides
by HCD versus 3907 peptides by UVPD), whereas UVPD identified greater
numbers of peptides for LysC digests (1033 peptides by UVPD versus
844 HCD), chymotrypsin digests (3219 peptides for UVPD versus 2921
for HCD), and GluC digests (2834 peptides for UVPD and 2393 for HCD)
and correspondingly greater numbers of proteins.